DESCRIPTION: (Applicant's Abstract) This project is aimed at a greater understanding of the mechanisms of regulation of neurotransmitter release, which is critical to an understanding of the function of the nervous system in health and disease. It uses a novel vertebrate preparation: the synapses formed by motoneuron neurites on muscle cells in Xenopus nerve-muscle cultures, in which pre- and postsynaptic processes can be patched-clamped and ionic currents analyzed directly and correlated with quantal neurotransmitter release. Of particular interest is the mechanism whereby Ca++ influx triggers vesicle fusion. Ca++ dynamics at active zones is the subject of many mathematical models, but direct measurements have proved difficult. In the Xenopus synapses, large-conductance Ca++-dependent K+ (KCa) channels are functionally coupled to Ca channels and probably concentrated at active zones. KCa currents will be used to monitor the concentration of Ca++ that occurs near them when Ca channels open during step depolarizations and action potentials. Whole-cell and single channel recordings will be used to calibrate the IKCa as a function of Ca++ concentration and voltage, and the IKCa will be used to describe the changes in Ca++ concentration that occur at or near active zones during different forms of synaptic activity. The changes will be correlated with the levels and timing of transmitter release. These data will be used to construct a realistic mathematical model of the Ca++ dynamics at active zones, which will be further refined by a characterization of the intrinsic buffers and experiments to assess the distance of release sites from Ca channels. We will also investigate the mechanisms of action of peptide fragments of the presynaptic proteins NSF and synapsin, which slow the kinetics of release in squid. We will investigate whether these peptides have the same effect in Xenopus and if they act by desynchronizing fusion events or slowing the kinetics of fusion.